Biochemistry and genetics of the ORMDL regulators of sphingolipid biosynthesis

鞘脂生物合成 ORMDL 调节因子的生物化学和遗传学

• 批准号: 9323552
• 负责人: BRIAN W. WATTENBERG
• 金额: $ 38.13万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9323552
• 关键词: Address; Affect; Agonist; Alpha Cell; Anabolism; Architecture; Asthma; Binding; Binding Sites; Biochemistry; CRISPR/Cas technology; Cardiovascular Diseases; Cell Line; Cell membrane; Cell-Free System; Cells; Ceramides; Childhood Asthma; Complex; Conserved Sequence; Detergents; Disease; Elements; Endoplasmic Reticulum; Enzymes; Epithelial; Genes; Genetic; Goals; Homologous Gene; Individual; Inflammatory; Knowledge; Lipids; Malignant Neoplasms; Measures; Mediating; Membrane; Membrane Proteins; Metabolic; Metabolism; Molecular; Mutation; Pathway interactions; Phenotype; Population Study; Protein Isoforms; Regulation; Research; Risk; Signal Transduction; Single Nucleotide Polymorphism; Smooth Muscle Myocytes; Specificity; Sphingolipids; Stimulus; Suggestion; System; Technology; Testing; Yeasts; asthmatic; cell type; eosinophil; genome editing; genome wide association study; novel; physical property; reconstitution; response; risk variant; serine palmitoyltransferase; tool

Sphingolipids are a diverse but metabolically related group of lipids with unique functional and physical properties that are critical for cell signaling and membrane architecture. Misregulation of particular sphingolipids has been implicated in such diverse diseases as cancer, inflammatory disease, and cardiovascular disease. In particular, as outlined below, genome-wide association studies have strongly implicated the subject of this proposal, the sphingolipid metabolic regulator ORMDL, in the risk for childhood asthma. The overall level of cellular sphingolipid is determined by the initiating, and rate limiting enzyme in the biosynthetic pathway, serine palmitoyltransferase (SPT). Not surprisingly, SPT activity is homeostatically controlled-i.e. SPT activity is strongly inhibited by elevated levels of cellular sphingolipid. We have demonstrated that this homeostatic control is mediated by a set of small membrane proteins found in the endoplasmic reticulum, the ORMDLs. The goal of the studies proposed here is to establish the mechanism by which the ORMDLs mediate SPT inhibition in response to elevated sphingolipid levels and to begin to uncover how those mechanisms impact on risk for asthma. These studies will answer basic questions concerning how ORMDLs sense cellular sphingolipid levels and how changes in sphingolipid levels are transmitted through ORMDL to affect SPT activity. We will test the hypothesis that ceramide directly binds ORMDL to trigger SPT regulation. We will determine the structural elements in ORMDL that are essential for mediating sphingolipid regulation of SPT, will use a recently constructed cell-free system to quantitate the levels of sphingolipid sensed by the ORMDL system, and will test the hypothesis that elevation of sphingolipid induces changes in ORMDL topology. Finally we will construct a reconstituted system with purified ORMDL and SPT to test the hypothesis that the ORMDLs and SPT are the only required components of this regulatory system. In addition we propose to address an essential question concerning the genetics of ORMDL expression that has emerged from genome-wide association studies in asthma. Single nucleotide polymorphisms (SNPs) adjacent to the gene of one of the ORMDL isoforms, ORMDL3, are strongly and consistently associated with the risk for childhood asthma and with elevated expression of ORMDL3. Although the population-based studies are intriguing and suggestive, a detailed examination of this phenomenon has been hamstrung by the inability to directly test the effect of these SNPs on ORMDL3 expression. It has not been possible to directly measure the degree to which these SNPs affect ORMDL3 expression nor in what cell types and conditions this elevated expression occurs. Here we propose to use emerging Crispr/Cas9 genome editing technology to produce paired cell lines, in asthma-relevant cell types, containing either a risk or non-risk genotype at the most prominent risk-associated SNP. These will be used to examine the extent, cell type, and stimuli specificity of elevation of ORMDL3 expression induced by the risk allele and how this affects sphingolipid metabolism.

鞘脂是一组多样化但代谢相关的脂质，具有独特的功能和物理特性 对于细胞信号传导和膜结构至关重要的特性。特定的监管不当 鞘脂与多种疾病有关，例如癌症、炎症性疾病和 心血管疾病。特别是，如下所述，全基因组关联研究强烈表明 暗示该提案的主题，鞘脂代谢调节剂 ORMDL，与儿童风险有关 哮喘。细胞鞘脂的总体水平由起始酶和限速酶决定 生物合成途径，丝氨酸棕榈酰转移酶（SPT）。毫不奇怪，SPT 活动是稳态的 受控——即SPT 活性受到细胞鞘脂水平升高的强烈抑制。我们有 证明这种稳态控制是由一组在细胞中发现的小膜蛋白介导的 内质网，ORMDL。这里提出的研究的目标是通过以下方式建立机制： ORMDL 响应鞘脂水平升高而介导 SPT 抑制，并开始揭示 这些机制如何影响哮喘风险。这些研究将回答有关如何 ORMDL 感知细胞鞘脂水平以及鞘脂水平的变化如何通过 ORMDL 影响 SPT 活动。我们将测试神经酰胺直接结合 ORMDL 以触发 SPT 的假设 规定。我们将确定 ORMDL 中对于介导鞘脂至关重要的结构元件 SPT 的调节，将使用最近构建的无细胞系统来定量鞘脂水平 由 ORMDL 系统感知，并将检验鞘脂升高引起变化的假设 ORMDL 拓扑。最后，我们将使用纯化的 ORMDL 和 SPT 构建重构系统来测试 假设 ORMDL 和 SPT 是该监管系统唯一必需的组成部分。此外 我们建议解决一个关于已经出现的 ORMDL 表达遗传学的基本问题 来自哮喘的全基因组关联研究。邻近的单核苷酸多态性 (SNP) ORMDL 同种型之一 ORMDL3 的基因与以下风险密切相关： 儿童哮喘且 ORMDL3 表达升高。尽管基于人群的研究 有趣且具有启发性的是，对这一现象的详细研究由于无法 直接测试这些 SNP 对 ORMDL3 表达的影响。目前还无法直接测量 这些 SNP 影响 ORMDL3 表达的程度以及这种升高的细胞类型和条件 表达发生。在这里我们建议使用新兴的 Crispr/Cas9 基因组编辑技术来生产 哮喘相关细胞类型中的配对细胞系，最多包含风险或非风险基因型 显着的风险相关 SNP。这些将用于检查的程度、细胞类型和刺激特异性 风险等位基因诱导的 ORMDL3 表达升高及其如何影响鞘脂代谢。